The importance of DNA based therapeutics, in particular in gene therapy, has led to increased research and development in this area (see, for example, Friedmann, T. Science, 1989, 244, 1275; Miller, A. D. Nature, 1992, 260, 455; Mulligan, R. C. Science, 1993, 260, 926; Wilson, J. M. Nature, 1993, 365, 691; Crystal, R. G. Nature Med., 1995, 1, 15). The use of these therapeutics can be problematic, however, because during drug delivery the DNA is subject to degradation. To maximize the power of these agents, it would be desirable to develop a mode of delivery in which the DNA-based therapeutic is protected from degradation.
Towards this end, several nano- or micro-encapsulation techniques have been developed and have been described in the literature (see, for example, Langer, R. S. Science 1990, 249, 1527; Kato et al. J Biol. Chem. 1991, 266, 3361; Jong et al., J. Controlled Release 1997, 47, 123; Mathiowitz et al., Nature 1997, 386, 410; Smith et al. Adv. Drug Del. Rev. 1997, 26, 135). Additionally, U.S. Pat. No. 5,407,609 by Tice et al. describes a method of microencapsulating biological or immunological agents to form a microencapsulated product. More recently, Ciftci et al. have developed a method to introduce DNA into mammalian cells using a polymer based gene delivery system. (Ciftci et al., Pharmaceutical Res. 1997, 14, s-639) This method, however only results in an encapsulation efficiency of 33-49%.
In particular, one of the most common techniques for preparation of biodegradable polymer microspheres encapsulating hydrophilic molecules is the double-emulsion solvent evaporation method. Using this technique, the molecule to be encapsulated is placed in aqueous solution while the polymer is dissolved in an organic phase commonly consisting of methylene chloride or ethyl acetate. The two phases (volume organic/volume aqueous=3-20) are emulsified, typically by sonication or homogenization. This primary emulsion is then added to a second aqueous phase and again mixed by homogenization to form the (water-in-oil)-in-water double emulsion. Upon evaporation of the partially water-miscible solvent, the polymer-containing droplets harden to form microspheres which can then be isolated by filtration or centrifugation. Lyophilization removes water from the interior aqueous phase resulting in a dry suspension of the encapsulated material within the polymer matrix. Unfortunately, however, the encapsulation efficiency of DNA into the hydrophobic matrix of PLGA was low (.about.20%) using this method. Additionally, the use of this method leads to a tendency of plasmid DNA to be converted from its supercoiled state to a nicked or linear state. The preservation of the supercoiled DNA is important because it is known that supercoiled DNA retains the highest level of bioactivity (Xu et al., Biochem. 1996, 35, 5616; Yamaizumi et al., Mol. Cell Biol. 1983, 3, 511).
Clearly, many of the methods described above still present a problem for DNA therapeutics because of the tendency of DNA therapeutics to degrade during and after the encapsulation process. Specifically, DNA stress induced degradation is encountered during homogenization and lyophilization. Furthermore, the DNA is susceptible to diffusing out of the aqueous phase, thus decreasing the encapsulation efficiency. Therefore, a method of encapsulating DNA based therapeutics that retains the integrity of the DNA (maximizes the supercoiled-DNA content) and increases the encapsulation efficiency would be desirable.